Recombinant DNA technology has been used to produce a variety of proteins of therapeutic or other economic value, including enzymes, growth factors and peptide hormones. These proteins have been produced in bacteria, fungal cells and, more recently, cultured mammalian cells. Because single-celled organisms cannot correctly process many human proteins, it has often been necessary to use cultured mammalian cells to make these proteins.
Mammalian cells can be transfected to express cloned DNA by well-established laboratory procedures. However, not all mammalian cell types will efficiently express transfected DNA sequences, and cells which have been shown to be efficient expressers of one transfected sequence will in other cases produce only low levels of other gene products. Low expression levels for transfected genes may be associated with degradation of the protein product intra- and/or extra-cellularly, production of inactive form(s) of the protein or production of form(s) of the protein that are cytotoxic. Low levels of protein or activity may also result from an unstable mRNA sequence, proteolytic activation or inadequate, inefficient or improper processing by the host cell. Processing steps which may be necessary for the activity or secretion of a newly synthesized protein include specific proteolytic cleavage, subunit polymerization, disulfide bond formation, post-translational or co-translational modification of certain amino acids and glycosylation.
These problems in protein production reflect the specialized nature of cells derived from higher organisms. Mammalian cells derived from a particular tissue may not properly produce a protein not normally made by that tissue. In addition, mammalian cell lines adapted to grow in culture are derived from tumors or are otherwise abnormal, often leading to unpredictable protein processing. For example, a number of research groups have produced human coagulation factor IX in cultured mammalian cells (Kaufman et al., J. Biol. Chem. 261:9622-9628, 1986; Anson et al., Nature 315:683-685, 1985; Hagen et al., EP 200,421; Busby et al., Nature 316:271-273, 1985). Despite efforts to maximize production of biologically active protein through the use of strong promoters, enhancers, increased gene copy number, etc., and despite the relatively high levels of factor IX mRNA observed, levels of active factor IX produced by these transfected cell lines do not exceed about 5 .mu.g/ml of cell culture medium. In some cases, precursor forms of factor IX are made but mature protein is ineffectively secreted from the host cell.
Problems with protein production have previously been dealt with by experimenting with a number of different cell types and by selecting and screening a large number of isolates of a particular transformed or transfected strain or cell line. Such an approach is extremely labor intensive and carries no assurance of success. Consequently, there is a need in the art for a method of systematically and predictably producing recombinant cells which can express proteins of interest in an active form and in economically feasible amounts. The present invention provides such a system, and further provides other related advantages.